Influenza virus infection enhances tumour-specific CD8+ T-cell immunity, facilitating tumour control.

Influenza A virus (IAV) can cause severe respiratory infection leading to significant global morbidity and mortality through seasonal epidemics. Likewise, the constantly increasing number of cancer diseases is a growing problem. Nevertheless, the understanding of the mutual interactions of the immune responses between cancer and infection is still very vague. Therefore, it is important to understand the immunological cross talk between cancer and IAV infection. In several preclinical mouse models of cancer, including melanoma and colorectal cancer, we observed that IAV infection in the lung significantly decreased the tumour burden. Concomitantly, tumour-specific CD8+ T-cells are strongly activated upon infection, both in the tumour tissue and in the lung. CD8+ T-cell depletion during infection reverses the reduced tumour growth. Interestingly, IAV infection orchestrated the migration of tumour-specific CD8+ T-cells from the tumour into the infected lung. Blocking the migration of CD8+ T-cells prevented the anti-tumoural effect. Thus, our findings show that viral respiratory infection has significant impact on the anti-tumour CD8+ T-cell response, which will significantly improve our understanding of the immunological cross talk between cancer and infection.

CD47 restricts antiviral function of alveolar macrophages during influenza virus infection.

CD47 is an ubiquitously expressed surface molecule with significant impact on immune responses. However, its role for antiviral immunity is not fully understood. Here, we revealed that the expression of CD47 on immune cells seemed to disturb the antiviral immune response as CD47-deficient mice (CD47-/-) showed an augmented clearance of influenza A virus (IAV). Specifically, we have shown that enhanced viral clearance is mediated by alveolar macrophages (aMФ). Although aMФ displayed upregulation of CD47 expression during IAV infection in wildtype mice, depletion of aMФ in CD47-/- mice during IAV infection reversed the augmented viral clearance. We have also demonstrated that CD47 restricts hemoglobin (HB) expression in aMФ after IAV and severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection, with HB showing antiviral properties by enhancing the IFN-ß response. Our study showed a negative role for CD47 during antiviral immune responses in the lung by confining HB expression in aMФ.

A Combination of Anti-PD-L1 Treatment and Therapeutic Vaccination Facilitates Improved Retroviral Clearance via Reactivation of Highly Exhausted T Cells.

PD-1-targeted therapies have shown modest antiviral effects in preclinical models of chronic viral infection. Thus, novel therapy protocols are necessary to enhance T cell immunity and viral control to overcome T cell dysfunction and immunosuppression. Here, we demonstrate that nanoparticle-based therapeutic vaccination improved PD-1-targeted therapy during chronic infection with Friend retrovirus (FV). Prevention of inhibitory signals by blocking PD-L1 in combination with therapeutic vaccination with nanoparticles containing the microbial compound CpG and a CD8+ T cell Gag epitope peptide synergistically enhanced functional virus-specific CD8+ T cell responses and improved viral clearance. We characterized the CD8+ T cell populations that were affected by this combination therapy, demonstrating that new effector cells were generated and that exhausted CD8+ T cells were reactivated at the same time. While CD8+ T cells with high PD-1 (PD-1hi) expression turned into a large population of granzyme B-expressing CD8+ T cells after combination therapy, CXCR5-expressing follicular cytotoxic CD8+ T cells also expanded to a high degree. Thus, our study describes a very efficient approach to enhance virus control and may help us to understand the mechanisms of combination immunotherapy reactivating CD8+ T cell immunity. A better understanding of CD8+ T cell immunity during combination therapy will be important for developing efficient checkpoint therapies against chronic viral infections and cancer.IMPORTANCE Despite significant efforts, vaccines are not yet available for every infectious pathogen, and the search for a protective approach to prevent the establishment of chronic infections, i.e., with HIV, continues. Immune checkpoint therapies targeting inhibitory receptors, such as PD-1, have shown impressive results against solid tumors. However, immune checkpoint therapies have not yet been licensed to treat chronic viral infections, since a blockade of inhibitory receptors alone provides only limited benefit, as demonstrated in preclinical models of chronic viral infection. Thus, there is a high interest in the development of potent combination immunotherapies. Here, we tested whether the combination of a PD-L1 blockade and therapeutic vaccination with functionalized nanoparticles is a potent therapy during chronic Friend retrovirus infection. We demonstrate that the combination therapy induced a synergistic reinvigoration of the exhausted virus-specific CD8+ T cell immunity. Taken together, our results provide further information on how to improve PD-1-targeted therapies during chronic viral infection and cancer.

Immunotherapy With Interferon α11, But Not Interferon Beta, Controls Persistent Retroviral Infection.

Type I Interferons (IFNs), including numerous IFNα subtypes and IFNß, are key molecules during innate and adaptive immune responses against viral infections. These cytokines exert various non-redundant biological activities, although binding to the same receptor. Persistent viral infections are often characterized by increased IFN signatures implicating a potential role of type I IFNs in disease pathogenesis. Using the well-established Friend retrovirus (FV) mouse model, we compared the therapeutic efficacy of IFNα11 and IFNß in acute and chronic retroviral infection. We observed a strong antiviral activity of both IFNs during acute FV infection, whereas only IFNα11 and not IFNß could also control persistent FV infection. The therapeutic treatment with IFNα11 induced the expression of antiviral IFN-stimulated genes (ISG) and improved cytotoxic T cell responses. Finally, dysfunctional CD8+ T cells solely regained cytotoxicity after IFNα11 treatment. Our data provide evidence for opposing activities of type I IFNs during chronic retroviral infections. IFNß was shown to be involved in immune dysfunction in chronic infections, whereas IFNα11 had a strong antiviral potential and reactivated exhausted T cells during persistent retroviral infection. In contrast, during acute infection, both type I IFNs were able to efficiently suppress FV replication.

In vivo biodistribution of calcium phosphate nanoparticles after intravascular, intramuscular, intratumoral, and soft tissue administration in mice investigated by small animal PET/CT.

Calcium phosphate nanoparticles were covalently surface-functionalized with the ligand DOTA and loaded with the radioisotope 68Ga. The biodistribution of such 68Ga-labelled nanoparticles was followed in vivo in mice by positron emission tomography in combination with computer tomography (PET-CT). The biodistribution of 68Ga-labelled nanoparticles was compared for different application routes: intravenous, intramuscular, intratumoral, and into soft tissue. The particle distribution was measured in vivo by PET-CT after 5 min, 15 min, 30 min, 1 h, 2 h, and 4 h, and ex vivo after 5 h. After intravenous injection (tail vein), the nanoparticles rapidly entered the lungs with later redistribution into liver and spleen. The nanoparticles remained mostly at the injection site following intramuscular, intratumoral, or soft tissue application, with less than 10 percent being mobilized into the blood stream. STATEMENT OF SIGNIFICANCE: The in vivo biodistribution of DOTA-terminated calcium phosphate nanoparticles was followed by PET/CT. To our knowledge, this is the first study of this kind. Four different application routes of clinical relevance were pursued: Intravascular, intramuscular, intratumoral, and into soft tissue. Given the high importance of calcium phosphate as biomaterial and for nanoparticular drug delivery and immunization, this is most important to assess the biofate of calcium phosphate nanoparticles for therapeutic application and also judge biodistribution of nanoscopic calcium phosphate ceramics, including debris from endoprostheses and related implants.

Combination immunotherapy with anti-PD-L1 antibody and depletion of regulatory T cells during acute viral infections results in improved virus control but lethal immunopathology.

Combination immunotherapy (CIT) is currently applied as a treatment for different cancers and is proposed as a cure strategy for chronic viral infections. Whether such therapies are efficient during an acute infection remains elusive. To address this, inhibitory receptors were blocked and regulatory T cells depleted in acutely Friend retrovirus-infected mice. CIT resulted in a dramatic expansion of cytotoxic CD4+ and CD8+ T cells and a subsequent reduction in viral loads. Despite limited viral replication, mice developed fatal immunopathology after CIT. The pathology was most severe in the gastrointestinal tract and was mediated by granzyme B producing CD4+ and CD8+ T cells. A similar post-CIT pathology during acute Influenza virus infection of mice was observed, which could be prevented by vaccination. Melanoma patients who developed immune-related adverse events under immune checkpoint CIT also presented with expanded granzyme-expressing CD4+ and CD8+ T cell populations. Our data suggest that acute infections may induce immunopathology in patients treated with CIT, and that effective measures for infection prevention should be applied.

Effective Activation of Human Antigen-Presenting Cells and Cytotoxic CD8+ T Cells by a Calcium Phosphate-Based Nanoparticle Vaccine Delivery System.

The ability of vaccines to induce T cell responses is crucial for preventing diseases caused by viruses. Nanoparticles (NPs) are considered to be efficient tools for the initiation of potent immune responses. Calcium phosphate (CaP) NPs are a class of biodegradable nanocarriers that are able to deliver immune activating molecules across physiological barriers. Therefore, the aim of this study was to assess whether Toll-like receptor (TLR) ligand and viral antigen functionalized CaP NPs are capable of inducing efficient maturation of human antigen presenting cells (APC). To achieve this, we generated primary human dendritic cells (DCs) and stimulated them with CpG or poly(I:C) functionalized CaP NPs. DCs were profoundly stronger when activated upon NP stimulation compared to treatment with soluble TLR ligands. This is indicated by increased levels of costimulatory molecules and the secretion of proinflammatory cytokines. Consequently, coculture of NP-stimulated APCs with CD8+ T cells resulted in a significant expansion of virus-specific T cells. In summary, our data suggest that functionalized CaP NPs are a suitable tool for activating human virus-specific CD8+ T cells and may represent an excellent vaccine delivery system.

A Tumor-Peptide-Based Nanoparticle Vaccine Elicits Efficient Tumor Growth Control in Antitumor Immunotherapy.

Recognition of immunoactive oligonucleotides by the immune system, such as Toll-like receptor ligand CpG, leads to increased antibody and T-cell responses. Systemic application often results in unwanted generalized nonantigen-specific activation of the immune system. Nanoparticles are ideal carriers for small and large molecules. Recently, we have demonstrated that calcium phosphate (CaP) nanoparticles functionalized with CpG, and viral antigens are able to induce specific T-cell immunity that protects mice against viral infection and efficiently reactivates the exhausted CD8+ T-cell compartment during chronic retroviral infection. Therefore, CaP nanoparticles are promising vaccine vehicles for therapeutic applications. In this study, we investigated the therapeutic potential use of these nanoparticles in a murine xenograft colorectal cancer model. Therapeutic vaccination with CaP nanoparticles functionalized with CpG and tumor model antigens increased the frequencies of cytotoxic CD8+ T cells in the tumor in a type I interferon-dependent manner. This was accompanied with significantly repressed tumor growth in contrast to the systemic administration of soluble CpG and antigens. Combination therapy of CaP nanoparticles and immune checkpoint blocker against PD-L1 further enhanced the cytotoxic CD8+ T-cell response and eradicated the tumors. Strikingly, vaccination with CaP nanoparticles functionalized with CpG and a primary tumor cell lysate was also sufficient to control the tumor growth. In conclusion, our results represent a translational approach for the use of CaP nanoparticles as a potent cancer vaccine vehicle.

Induction of Type I Interferons by Therapeutic Nanoparticle-Based Vaccination Is Indispensable to Reinforce Cytotoxic CD8+ T Cell Responses During Chronic Retroviral Infection.

T cell dysfunction and immunosuppression are characteristic for chronic viral infections and contribute to viral persistence. Overcoming these burdens is the goal of new therapeutic strategies to cure chronic infectious diseases. We recently described that therapeutic vaccination of chronic retrovirus infected mice with a calcium phosphate (CaP) nanoparticle (NP)-based vaccine carrier, functionalized with CpG and viral peptides is able to efficiently reactivate the CD8+ T cell response and improve the eradication of virus infected cells. However, the mechanisms underlying this effect were largely unclear. While type I interferons (IFNs I) are considered to drive T cell exhaustion by persistent immune activation during chronic viral infection, we here describe an indispensable role of IFN I induced by therapeutic vaccination to efficiently reinforce cytotoxic CD8+ T cells (CTL) and improve control of chronic retroviral infection. The induction of IFN I is CpG dependent and leads to significant IFN signaling indicated by upregulation of IFN stimulated genes. By vaccinating chronically retrovirus-infected mice lacking the IFN I receptor (IFNAR-/-) or by blocking IFN I signaling in vivo during therapeutic vaccination, we demonstrate that IFN I signaling is necessary to drive full reactivation of CTLs. Surprisingly, we also identified an impaired suppressive capability of regulatory T cells in the presence of IFNα, which implicates an important role for vaccine-induced IFNα in the regulation of the T cell response during chronic retroviral infection. Our data suggest that inducing IFN I signaling in conjunction with the presentation of viral antigens can reactivate immune functions and reduce viral loads in chronic infections. Therefore, we propose CaP NPs as potential therapeutic tool to treat chronic infections.

Delivery of the TLR ligand poly(I:C) to liver cells in vitro and in vivo by calcium phosphate nanoparticles leads to a pronounced immunostimulation.

The selective activation of the immune system is a concurrent problem in the treatment of persistent diseases like viral infections (e.g. hepatitis). For the delivery of the toll-like receptor ligand poly(I:C), an immunostimulatory action was discovered earlier by hydrodynamic injection. However, this technique is not clinically transferable to human patients. A modular system where the immunoactive toll-like-receptor ligand 3 (TLR-3) poly(I:C) was incorporated into calcium phosphate nanoparticles was developed. The nanoparticles had a hydrodynamic diameter of 275nm and a zeta potential of +20mV, measured by dynamic light scattering. The diameter of the solid core was 120nm by scanning electron microscopy. In vitro, the nanoparticle uptake was investigated after 1 and 24h of incubation of THP-1 cells (macrophages) with nanoparticles by fluorescence microscopy. After intravenous injection into BALB/c and C57BL/6J mice, respectively, the in vivo uptake was especially prominent in lung and liver, 1 and 3h after the injection. Pronounced immunostimulatory effects of the nanoparticles were found in vitro with primary liver cells, i.e. Kupffer cells (KC) and liver sinusoidal endothelial cells (LSEC) from wild-type C57BL/6J mice. Thus, they represent a suitable alternative to hydrodynamic injection treatments for future vaccination concepts. STATEMENT OF SIGNIFICANCE: The selective activation of the immune system is a concurrent problem in the treatment of persistent diseases like viral infections (e.g. hepatitis). For the delivery of the toll-like receptor ligand poly(I:C), an immunostimulatory action has been discovered earlier by hydrodynamic injection. However, this technique is not clinically transferable to human patients. We have developed a modular system where poly(I:C) was incorporated into calcium phosphate nanoparticles. The uptake into relevant liver cells was studied both in vitro and in vivo. After intravenous injection into mice, the in vivo uptake was especially prominent in lung and liver, 1 and 3h after the injection. The corresponding strong immune reaction proves their high potential to turn up the immune system, e.g. against viral infections, without adverse side reactions.

Local delivery of siRNA-loaded calcium phosphate nanoparticles abates pulmonary inflammation.

The local interference of cytokine signaling mediated by siRNA-loaded nanoparticles might be a promising new therapeutic approach to dampen inflammation during pulmonary diseases. For the local therapeutic treatment of pulmonary inflammation, we produced multi-shell nanoparticles consisting of a calcium phosphate core, coated with siRNAs directed against pro-inflammatory mediators, encapsulated into poly(lactic-co-glycolic acid), and coated with a final outer layer of polyethylenimine. Nasal instillation of nanoparticles loaded with a mixture of siRNAs directed against different cytokines to mice suffering from TH1 cell-mediated lung inflammation, or of siRNA directed against NS-1 in an influenza infection model led to a significant reduction of target gene expression which was accompanied by distinct amelioration of lung inflammation in both models. Thus, this study provides strong evidence that the specific and local modulation of the inflammatory response by CaP/PLGA nanoparticle-mediated siRNA delivery could be a promising approach for the treatment of inflammatory disorders of the lung.

Avidin-conjugated calcium phosphate nanoparticles as a modular targeting system for the attachment of biotinylated molecules in vitro and in vivo.

Avidin was covalently conjugated to the surface of calcium phosphate nanoparticles, coated with a thin silica shell and terminated by sulfhydryl groups (diameter of the solid core about 50nm), with a bifunctional crosslinker connecting the amino groups of avidin to the sulfhydryl group on the nanoparticle surface. This led to a versatile nanoparticle system where all kinds of biotinylated (bio-)molecules can be easily attached to the surface by the non-covalent avidin-biotin-complex formation. It also permits the attachment of different biomolecules on the same nanoparticle (heteroavidity), creating a modular system for specific applications in medicine and biology. The variability of the binding to the nanoparticle surface of the was demonstrated with various biotinylated molecules, i.e. fluorescent dyes and antibodies. The accessibility of the conjugated avidin was demonstrated by a fluorescence-quenching assay. About 2.6 binding sites for biotin were accessible on each avidin tetramer. Together with a number of about 240 avidin tetramer units per nanoparticle, this offers about 600 binding sites for biotin on each nanoparticle. The uptake of fluorescently labelled avidin-conjugated calcium phosphate nanoparticles by HeLa cells showed the co-localization of fluorescent avidin and fluorescent biotin, indicating the stability of the complex under cell culture conditions. CD11c-antibody functionalized nanoparticles specifically targeted antigen-presenting immune cells (dendritic cells; DCs) in vitro and in vivo (mice) with high efficiency. STATEMENT OF SIGNIFICANCE: Calcium phosphate nanoparticles have turned out to be very useful transporters for biomolecules into cells, both in vitro and in vivo. However, their covalent surface functionalization with antibodies, fluorescent dyes, or proteins requires a separate chemical synthesis for each kind of surface molecule. We have therefore developed avidin-terminated calcium phosphate nanoparticles to which all kinds of biotinylated molecules can be easily attached, also as a mixture of two or more molecules. This non-covalent bond is stable both in cell culture and after injection into mice in vivo. Thus, we have created a highly versatile system for many applications, from the delivery of biomolecules over the targeting of cells and tissue to in vivo imaging.

Combination of nanoparticle-based therapeutic vaccination and transient ablation of regulatory T cells enhances anti-viral immunity during chronic retroviral infection.

BACKGROUND: Regulatory T cells (Tregs) have been shown to limit anti-viral immunity during chronic retroviral infection and to restrict vaccine-induced T cell responses. The objective of the study was to assess whether a combinational therapy of nanoparticle-based therapeutic vaccination and concomitant transient ablation of Tregs augments anti-viral immunity and improves virus control in chronically retrovirus-infected mice. Therefore, chronically Friend retrovirus (FV)-infected mice were immunized with calcium phosphate (CaP) nanoparticles functionalized with TLR9 ligand CpG and CD8(+) or CD4(+) T cell epitope peptides (GagL85-93 or Env gp70123-141) of FV. In addition, Tregs were ablated during the immunization process. Reactivation of CD4(+) and CD8(+) effector T cells was analysed and the viral loads were determined. RESULTS: Therapeutic vaccination of chronically FV-infected mice with functionalized CaP nanoparticles transiently reactivated cytotoxic CD8(+) T cells and significantly reduced the viral loads. Transient ablation of Tregs during nanoparticle-based therapeutic vaccination strongly enhanced anti-viral immunity and further decreased viral burden. CONCLUSION: Our data illustrate a crucial role for CD4(+) Foxp3(+) Tregs in the suppression of anti-viral T cell responses during therapeutic vaccination against chronic retroviral infection. Thus, the combination of transient Treg ablation and therapeutic nanoparticle-based vaccination confers robust and sustained anti-viral immunity.

Virus-Induced Type I Interferon Deteriorates Control of Systemic Pseudomonas Aeruginosa Infection.

BACKGROUND: Type I interferon (IFN-I) predisposes to bacterial superinfections, an important problem during viral infection or treatment with interferon-alpha (IFN-α). IFN-I-induced neutropenia is one reason for the impaired bacterial control; however there is evidence that more frequent bacterial infections during IFN-α-treatment occur independently of neutropenia. METHODS: We analyzed in a mouse model, whether Pseudomonas aeruginosa control is influenced by co-infection with the lymphocytic choriomeningitis virus (LCMV). Bacterial titers, numbers of neutrophils and the gene-expression of liver-lysozyme-2 were determined during a 24 hours systemic infection with P. aeruginosa in wild-type and Ifnar(-/-) mice under the influence of LCMV or poly(I:C). RESULTS: Virus-induced IFN-I impaired the control of Pseudomonas aeruginosa. This was associated with neutropenia and loss of lysozyme-2-expression in the liver, which had captured P. aeruginosa. A lower release of IFN-I by poly(I:C)-injection also impaired the bacterial control in the liver and reduced the expression of liver-lysozyme-2. Low concentration of IFN-I after infection with a virulent strain of P. aeruginosa alone impaired the bacterial control and reduced lysozyme-2-expression in the liver as well. CONCLUSION: We found that during systemic infection with P. aeruginosa Kupffer cells quickly controlled the bacteria in cooperation with neutrophils. Upon LCMV-infection this cooperation was disturbed.

Prophylactic and therapeutic vaccination with a nanoparticle-based peptide vaccine induces efficient protective immunity during acute and chronic retroviral infection.

Retroviral infections e.g. HIV still represent a unique burden in the field of vaccine research. A common challenge in vaccine design is to find formulations that create appropriate immune responses to protect against and/or control the given pathogen. Nanoparticles have been considered to be ideal vaccination vehicles that mimic invading pathogens. In this study, we present biodegradable calcium phosphate (CaP) nanoparticles, functionalized with CpG and retroviral T cell epitopes of Friend virus (FV) as excellent vaccine delivery system. CaP nanoparticles strongly increased antigen delivery to antigen-presenting cells to elicit a highly efficient T cell-mediated immune response against retroviral FV infection. Moreover, single-shot immunization of chronically FV-infected mice with functionalized CaP nanoparticles efficiently reactivated effector T cells which led to a significant decrease in viral loads. Thus, our findings clearly indicate that a nanoparticle-based peptide immunization is a promising approach to improve antiretroviral vaccination. FROM THE CLINICAL EDITOR: In this study, biodegradable calcium phosphate nanoparticles were used as a vaccine delivery system after functionalization with CpG and Friend virus-derived T-cell epitopes. This vaccination strategy resulted in increased T-cell mediated immune response even in chronically infected mice, providing a promising approach to the development of clinically useful antiretroviral vaccination strategies.

The type of adjuvant strongly influences the T-cell response during nanoparticle-based immunization.

Potent vaccines require the ability to effectively induce immune responses. Especially for the control of infectious diseases with intracellular pathogens, like viruses or bacteria, potent T-cell responses are indispensable. Several delivery systems such as nanoparticles have been considered to boost the immunogenicity of pathogen derived peptides or subunits for the induction of potent T-cell responses. Since they can be further functionalized with immunostimulants, like Toll-like receptor (TLR) agonists, they improve the response by enhanced activation of the innate immune system. Currently, TLR agonists like unmethylated CpG oligonucleotides and the synthetic dsRNA derivate polyriboinosinic acid-polyribocytidylic acid (poly[I:C]) are widely used as vaccine adjuvants. CpG and poly(I:C) trigger different TLRs and therefore show differential signal transduction. Recently, we established biodegradable calcium phosphate (CaP) nanoparticles as potent T cell inducing vaccination vehicles. In this commentary we discuss the role of CpG and poly(I:C) for the effective induction of virus-specific T cells during immunization with CaP nanoparticles. The presented results underline the importance of the right formulation of vaccines for specific immunization purpose.

Immunization with biodegradable nanoparticles efficiently induces cellular immunity and protects against influenza virus infection.

The ability of vaccines to induce T cell responses is crucial for preventing diseases caused by viruses or bacteria. Nanoparticles (NPs) are considered an efficient tool for inducing potent immune responses. In this study, we describe a novel vaccination approach with biodegradable calcium phosphate (CaP) NPs that serve as carrier of immunoactive TLR9 ligand (CpG) combined with a viral Ag from the influenza A virus hemagglutinin. Functionalized CaP NPs were efficiently taken up by dendritic cells in vivo and elicited a potent T cell-mediated immune response in immunized mice with high numbers of IFN-γ-producing CD4(+) and CD8(+) effector T cells. Most importantly, both i.p. and intranasal immunization with these NPs offered protection in a mouse model of influenza virus infection. This study demonstrates the great potential of CaP NPs as a novel vaccination tool that offers substantial flexibility for several infection models.

Mechanism of the uptake of cationic and anionic calcium phosphate nanoparticles by cells.

The uptake of calcium phosphate nanoparticles (diameter 120nm) with different charge by HeLa cells was studied by flow cytometry. The amount of uptaken nanoparticles increased with increasing concentration of nanoparticles in the cell culture medium. Several inhibitors of endocytosis and macropinocytosis were applied to elucidate the uptake mechanism of nanoparticles into HeLa cells: wortmannin, LY294002, nocodazole, chlorpromazine and nystatin. Wortmannin and LY294002 strongly reduced the uptake of anionic nanoparticles, which indicates macropinocytosis as uptake mechanism. For cationic nanoparticles, the uptake was reduced to a lesser extent, indicating a different uptake mechanism. The localization of nanoparticles inside the cells was investigated by conjugating them with the pH-sensitive dye SNARF-1. The nanoparticles were localized in lysosomes after 3h of incubation.

Generation and function of immunosuppressive human and murine CD8+ T cells by transforming growth factor-ß and retinoic acid.

The intestinal immune system is constantly challenged by foreign antigens and commensal bacteria. Therefore, proper control of the intestinal microenvironment is required. One important arm of this regulatory network consists of regulatory T cells. In contrast to CD4(+) Foxp3(+) regulatory T cells, which have been well characterized, immunomodulatory CD8(+) T cells that express Foxp3 are less well defined in terms of their generation and function. Failures of these regulatory mechanisms contribute to the development of inflammatory bowel disease. In this study we demonstrate that the frequency of CD8(+) Foxp3(+) T cells is reduced in the peripheral blood of patients with ulcerative colitis. As these cells might play a currently underestimated role in the maintenance of intestinal homeostasis, we have investigated human and murine CD8(+) Foxp3(+) T cells generated by stimulating naive CD8(+) T cells in the presence of transforming growth factor-ß and retinoic acid, mediators that are abundantly produced in the intestinal mucosa. These CD8(+) Foxp3(+) fully competent regulatory T cells show strong expression of regulatory molecules CD25, Gpr83 and CTLA-4 and exhibit cell-cell contact-dependent immunosuppressive activity in vitro. Our study illustrates a previously unappreciated critical role of CD8(+) Foxp3(+) T cells in controlling potentially dangerous T cells and in the maintenance of intestinal homeostasis.

CD8+ T-cell response promotes evolution of hepatitis C virus nonstructural proteins.

BACKGROUND & AIMS: Hepatitis C virus (HCV) acquires mutations that allow it to escape the CD8+ T-cell response, although the extent to which this process contributes to viral evolution at the population level is not clear. We studied viral adaptation using data from a large outbreak of HCV genotype 1b infection that occurred among women immunized with contaminated immunoglobulin from 1977 to 1978. METHODS: The HCV nonstructural protein coding regions NS3-NS5B were sequenced from 78 patients, and mutations were mapped according to their location inside or outside previously described CD8+ T-cell epitopes. A statistical approach was developed to identify sites/regions under reproducible selection pressure associated with HLA class I. RESULTS: The frequency of nonsynonymous mutations was significantly higher inside previously described CD8+ T-cell epitopes than outside-particularly in NS3/4A and NS5B. We identified new regions that are under selection pressure, indicating that not all CD8+ T-cell epitopes have been identified; 6 new epitopes that interact with CD8+ T cells were identified and confirmed in vitro. In some CD8+ T-cell epitopes mutations were reproducibly identified in patients that shared the relevant HLA allele, indicating immune pressure at the population level. There was statistical support for selection of mutations in 18 individual epitopes. Interestingly, 14 of these were restricted by HLA-B allele. CONCLUSIONS: HLA class I-associated selection pressure on the nonstructural proteins and here predominantly on NS3/4A and NS5B promotes evolution of HCV. HLA-B alleles have a dominant effect in this selection process. Adaptation of HCV to the CD8+ T-cell response at the population level creates challenges for vaccine design.